Spatial fragmentation in the distribution of diatom endosymbionts from the taxonomically clarified dinophyte Kryptoperidinium triquetrum (= Kryptoperidinium foliaceum, Peridiniales)

Among the photosynthetically active dinophytes, the Kryptoperidiniaceae are unique in having a diatom as endosymbiont instead of the widely present peridinin chloroplast. Phylogenetically, it is unresolved at present how the endosymbionts are inherited, and the taxonomic identities of two iconic dinophyte names, Kryptoperidinium foliaceum and Kryptoperidinium triquetrum, are also unclear. Multiple strains were newly established from the type locality in the German Baltic Sea off Wismar and inspected using microscopy as well as molecular sequence diagnostics of both host and endosymbiont. All strains were bi-nucleate, shared the same plate formula (i.e., po, X, 4′, 2a, 7′′, 5c, 7s, 5′′′, 2′′′′) and exhibited a narrow and characteristically L-shaped precingular plate 7′′. Within the molecular phylogeny of Bacillariaceae, endosymbionts were scattered over the tree in a highly polyphyletic pattern, even if they were gained from different strains of a single species, namely K. triquetrum. Notably, endosymbionts from the Baltic Sea show molecular sequences distinct from the Atlantic and the Mediterranean Sea, which is the first report of such a spatial fragmentation in a planktonic species of dinophytes. The two names K. foliaceum and K. triquetrum are taxonomically clarified by epitypification, with K. triquetrum having priority over its synonym K. foliaceum. Our study underlines the need of stable taxonomy for central questions in evolutionary biology.

x Figure S16 W1  www.nature.com/scientificreports/ of similar height, but the right lateral plate 6′′ was higher than the others (Fig. 4A,B,D,E). Plate 7′′ was conspicuously L-or boot-shaped with a narrow upper part and a broader base abutting C5. This plate always appeared very bright under fluorescent light of stained samples ( Fig. 4A-C), but SEM revealed no obvious difference in plate thickness or surface structure (Fig. 5B,D). The cingular groove was discontinuous and disconnected ventrally by plate 1′ p (Fig. 4A,B,I). Plates C1 and C2 were of similar size and smaller than the remaining cingular plates (Fig. 4A-F). The suture between plates C2 and C3 was in lateral position and thus often difficult to observe. In the hypotheca (Fig. 4A-F), plate 3′′′ was in dorsal position and abutted both antapical plates (Fig. 4D), which were of comparable size (Fig. 4A,B,D). The sulcal area was dominated by two large plates, the right and posterior sulcal plates sd and sp, respectively (Fig. 4A,B). Plate sd was roughly rectangular and abutted posteriorly the right side of the large and asymmetric www.nature.com/scientificreports/ plate sp. The left anterior side of plate sp was triangular and shared a broad suture with plate 1′′′ (Fig. 4A,B). The small plates in the central sulcal area were difficult to observe by LM, but two tongue-shaped plates (a posterior www.nature.com/scientificreports/ left sulcal plate: ssp and an anterior left sulcal plate: ssa) were clearly visible. Anteriorly to plate ssa, there was a small and posteriorly curved anterior sulcal plate sa contacting plates C1 and 1′ p (Fig. 4I). On the left side of the large right sulcal plate sd, there was an elongated anterior median sulcal plate sma, which always was brightly stained (Fig. 4A,B,I).
Using SEM (Figs. 5, 6), thecal pore size was estimated as 0.15-0.20 µm in diameter. A few plates were consistently free of pores, namely the pore plate, the X-plate (Fig. 5C) and all small central sulcal plates (Fig. 6). There was a dense row of pores on postcingular plates below the cingulum with its five cingular plates (Fig. 5F). Moreover, SEM enabled detailed observations of number and arrangement of the small plates in the central sulcus (Fig. 6). In a presumably undisturbed arrangement, plates sd and 1′′′ were in close proximity posteriorly to the flagellar pore region and formed a narrow, closed canal for the longitudinal flagellum (arrow in Figs. 2C,     (Fig. 6D,E,G). Exceptionally, this plate was artificially separated from plate sd and was seen on the left side still closely attached to plate ssa (Fig. 6D). In the central sulcal area, the larger and tongue-like posterior left sulcal plate ssp was visible. Between plates ssp and sma, there was another small and narrow sulcal plate, namely the posterior median sulcal plate smp, which was not clearly visible in LM (Fig. 4I).
On the other hand, the small anterior sulcal plate sa (anteriorly of the flagellar pore area) was clearly visible in LM (Fig. 4A,B,I) but was lost or could not be clearly observed in SEM preparations (Fig. 6A   The SSU + ITS + LSU + psbA + rbcL + pcbC alignment of diatoms was 1892 + 1221 + 3356 + 1005 + 1620 + 13 77 bp long and was composed of 545 + 797 + 494 + 219 + 603 + 459 parsimony-informative sites (30%, mean of 8.73 per terminal taxon) and 5417 distinct RAxML alignment patterns. Topological inconsistencies between nuclear and plastid loci were rare and-if present-referred to internal branching of, for example, Chaetoceros, Cylindrotheca and Pseudo-nitzschia. Figure 9 (Supplementary Figure 9) shows the best-scoring ML tree (− ln = 140,379.69), with many nodes having high if not maximal statistical support. Although some deeper nodes had only low support, Bacillariaceae (84LBS, 1.00BPP) were monophyletic with respect to the successive close relatives "Amphora" (84LBS, 1.00BPP), Naviculales (97LBS, 1.00BPP), Eunotia (100LBS, 1.00BPP) and Chaetoceros (100LBS, 1.00BPP). Dinophyte endosymbionts did not constitute a monophyletic group, with that of Blixaea nesting with Chaetoceros tenuissimus (100LBS) and those of Dinothrix, Durinskia and Kryptoperidinium scattered over the tree in a polyphyletic pattern.  Figure 9. A molecular reference tree recognising major groups of Bacillariaceae (created using Adobe Illustrator © CS6; https:// www. adobe. com/ de/ produ cts/ illus trator. html). Maximum Likelihood (ML) tree of 317 bacillariacean sequences (with strain number and GenBank accession number information, outgroup accessions are shaded grey) as inferred from an alignment comprising sequences of the rRNA operon, psbA, rbcL and psbC (3117 parsimony-informative positions). Clade labelling follows previous work 66 . Numbers on branches are ML bootstrap (above) and Bayesian probabilities (below) for the clusters (asterisks indicate maximal support values, values under 50 and .90, respectively, are not shown). Note that endosymbionts of Kryptoperidiniaceae (emphasised by red lettering) are scattered over the tree in a highly polyphyletic pattern, accessions assigned to Kryptoperidinium are indicated by pink arrows. Freshwater accessions are highlighted by green branches. www.nature.com/scientificreports/ Endosymbiont ITS sequences collected in the Baltic Sea were the same as each other but with two exceptions (i.e., W4-A6, W4-F1, from Wismar marina), which differed in a unique 5 bp insertion from the others. In the phylogenetic tree, endosymbionts of K. triquetrum were nested in clade 6B (51LBS) with other dinophyte endosymbionts, but comprised two clades, which were only distantly related to each other: Accessions from the Baltic Sea constituted a group (91LBS, 1.00BPP) with free-living species determined as "Nitzschia" lembiformis, "Nitzschia" pusilla and "Nitzschia" thermalis; accessions from the Atlantic Ocean and the Mediterranean Sea comprised a clade (97LBS, 1.00BPP) with predominantly freshwater taxa including "Nitzschia" draveillensis. The latter clade showed a close relationship (96LBS, 1.00BPP) to sequences retrieved from endosymbionts of Durinskia capensis (100LBS, 1.00BPP). Notably, the ITS sequence of strain GeoB 459 was almost identical (> 99% similarity) to an ITS sequence (AY574381) derived from free-living "Nitzschia" pusilla (89LBS, .91BPP).

Discussion
Repeated uptake of endosymbiont partners. The establishment of permanent chloroplast organelles in eukaryotic cells from formerly free-living cooperative partners is a multi-step process of evolution 3,5,6 . In Archaeplastida, the primary endosymbiosis event has resulted in a mutual dependence of the partners, which is absolute 67 -neither the chloroplasts nor the host cells are able to survive without each other under natural conditions 68 . Replication is also synchronised, and there has been an extensive exchange of genetic material between the compartments nucleus and plastid 69 . At the levels of secondary and tertiary endosymbiosis, the amount and maturity of such cooperation are highly diverse: Some species and species groups have already developed a similar dependency as in algae with primary endosymbiosis (e.g., cryptophytes 70 ), others are still at the dawn of such a progression 7 .
The present case of Kryptoperidinium as integral part of the dinotoms certainly represent an early stage of chloroplast establishment, and some of the multiple steps can be brought into a sequence of evolutionary events: Replication between hosts and diatoms appears already synchronised 53,71,72 , but almost intact cell anatomy of the endosymbionts is retained 23,35,39,57 , and genome reduction is still insignificant 27,[73][74][75] . Nevertheless, it has been suggested that the endosymbionts of Kryptoperidiniaceae are hosted permanently and inherited vertically after a single, ancient engulfment event 65,76,77 . If the chloroplasts are inherited vertically, then endosymbionts would form a monophyletic group in the trees derived from molecular sequence data (like chloroplasts nesting in cyanobacteria 12,13,15 ). However, the phylogenetic results clearly reject this hypothesis, and the opposite is the case: The endosymbionts are scattered over the tree, and most of them have closest relatives not among other endosymbionts but among free-living diatoms 7,41 . This conclusion does not only refer to groups of species but even to single species such as K. triquetrum, in which there are two distinct and only distantly related groups of endosymbionts in the bacillariacean tree.
The presence of different diatoms in the same host species indicates that tertiary endosymbiosis is not yet a stable system in Kryptoperidiniaceae, and the question arises whether the endosymbiosis is entirely obligate (or some individuals may be able to survive entirely heterotrophically, lacking any endosymbiont). Anyhow, recent work on Durinskia shows that endosymbiont establishment even at the species level may reflect different evolutionary stages 42 . One species, namely Duriskia capensis, keeps newly phagocytosed diatoms for only two months, whereas other species are able to maintain diatoms for undetermined periods of time. Strains assigned to Kryptoperidinium have kept their endosymbiont for more than 30 years in cultivation 78 . Nevertheless, cells of Kryptoperidiniaceae with only one stainable nucleus under light microscopy have been mentioned 36,52,64 , but such reports should be taken with reservation in Kryptoperidinium (not least because of the methodological challenges). All strains of K. triquetrum studied here are bi-nucleate and for the moment, the presence of the diatom nucleus is therefore considered an invariable trait of the species.

Distinct ribotypes of endosymbionts in different regions of the world. Plankton communities
may actually consist of both wide spread and more restrictedly distributed species and are assembled to a combination of dispersal potential and ecological selection 46,[79][80][81] . In a number of planktonic dinophytes such as Alexandrium (Ostreopsidaceae) and Scrippsiella (Thoracosphaeraceae), ITS ribotypes show a global distribution 81,82 . Benthic dinophytes do not show a clear signal, with ITS ribotypes of Coolia (Ostreopsidaceae) found worldwide 83 , whereas epiphytic Ostreopsis (also Ostreopsidaceae) in fact show a correlation between molecular sequence data and distribution, with genetically distinct Atlantic/Mediterranean versus Indo-Pacific populations 84 . In the freshwater environment, there may be some morphological differentiation within species, such as in Peridinium volzii between specimens from Europe and Eastern Asia 85 . In the present study, K. triquetrum does show a spatial distinction based on multiple gatherings, as the Baltic strains have different endosymbionts in this species than strains from other localities. To the best of our knowledge, this is the first report of such a spatial fragmentation in a planktonic species of dinophytes. It is worth noting again here that the endosymbionts of K. triquetrum do not constitute a monophyletic group, but have closest relatives among free-living diatoms.
The spatially regular meeting of the prospective partners is one of the prerequisites at the dawn of chloroplast establishment [3][4][5]7 . Most members of the Bacillariaceae are benthic algae, living on shallow marine sediments (but also as periphyton and epiliton), whereas dinotoms such as K. triquetrum are mainly planktonic forms 32,65 . How precisely a planktonic dinophyte would capture a benthic diatom remains a question for future research. It is currently still under debate whether endemism is an important phenomenon in benthic diatoms 86-88 -if restricted distribution patterns do occur, then the presence of different partners in hosts of different geographical origins would explain the present molecular trees of Bacillariaceae with the endosymbionts included.
Kryptoperidiniaceae are an exceptional model for studying the first steps of organelle establishment, as the excessive reduction of the morphological and biochemical components that has occurred in other photosynthetic groups has not yet taken place. However, research only begins to understand the complex interactions and mutual www.nature.com/scientificreports/ processes that have led to the diversity of photosynthesis in eukaryotes. In the case of the Kryptoperidiniaceae, evolutionary conclusions suffer from weakly supported phylogenies of the endosymbionts, and improved DNA trees of diatoms are needed. Concatenation of sequences 66,89,90 is still not universally accepted as the method to reach this aim (similar to the situation in dinophytes). The present attempt of this study follows this path (like it is done also in dinophytes 46,[91][92][93], although the alignment is still very patchy-these gaps need to be filled in future research. To robustly support the results of Kryptoperidinium shown here, multiple collections and strains of one species as well as of closely related species and populations are needed regarding both hosts and endosymbionts.

Divergent thecal interpretations.
Based on the observations of multiple strains from various geographic regions the morphology of accessions assigned to Kryptoperidinium I is very consistent, and we are confident that the lineage comprises a single species only (with K. foliaceum being a later heterotypic synonym of K. triquetrum). This conclusion enables a critical assessment of morphological inconsistencies that are found in the literature. With respect to the thecal plate pattern of Kryptoperidinium (Table 2), there is general consensus in the number of postcingular (i.e., five) and antapical plates (i.e., two) of the hypotheca (as frequently present in peridiniod dinophytes), but varying numbers of epithecal, cingular and sulcal plates have been encountered. However, the comparison of historical reports is hampered, because phylogenetic analyses indicate the existence of two, only distantly related clades of Kryptoperidinium 64 having similar appearance 43,52-54 . Unfortunately, most previous morphological studies lack corresponding molecular sequence data and hence, it is difficult to distin- Table 2. Plate patterns of Kryptoperidinium reported in the literature. a Among the 4 apical plates described and drawn, there is a symmetric first apical plate ('Rautenplatte' or 1r), which is considered rare. Based on the present observations the existence is doubtful. b In the drawing, the ventral precingular plate refers to our first apical plate leading to six precingular plates. The L-shaped precingular plate 7′′ was probably overlooked. c This plate was not labelled but considered as sulcal plate: "Längsfurche in der Form eines dreieckigen Feldes auf die Epivalva übergreifend" (translated: "sulcus with a triangular-shaped plate extends into the epitheca"). d There are three plates surrounding the APC (i.e., three apical plates), and the large ventral plate (usually considered as plate 1′) is interpreted as precingular plate. The elongated X-plate separating plate 1′ from the pore plate was obviously overlooked. e These seven plates include the ventral plate (1′), that six true precingular plates were observed. However, the L-shaped precingular plate 7′′ might have been overlooked. f "A small triangular plate dividing the ends of the girdle apparently belongs to the ventral area". g "In der Längsfurche befinden sich drei Platten, … während die dritte obere die nicht geschlossene Äquatorialfurche ergänzt und auch besonders an der linken Seite der Rautenplatte auf die Epivalva übergreift" (translated: "In the sulcus, there are three plates, … whereas the anterior third plate complete the cingulum and extends on the left side of plate 1′ into the epitheca"). h "Occasionally with four apicals". i Determined as Peridinium foliaceum. j The ventral area between the start and the end of the cingulum is formed by a "fermée par une plaque supplementaire" (translated: "supplementary plate"). k In his doctoral thesis, Takeo Horiguchi presented a plate pattern, for what he determined as "Glenodinium foliaceum" -stage of a G. foliaceum-Dinothrix paradoxa complex. These cells, however, differ fundamentally from K. foliaceum by a significant cingular displacement, by lack of dorso-ventral compression and by the presence of a symmetrical plate 1′ in mid-ventral position. l Determined as Peridinium foliaceum. m Among five strains, they described four strains with four and one strain with five cingular plates. n SSU and ITS data for three of the five studied strains available (i.e., NCMA1326, SC, UTEX1688), which all belong to Kryptoperidinium II. o As Kryptoperidinium sp. p There is no conclusive information, how this uncertainty is inferred or has to be interpreted. In Fig. 6D, four sulcal plates are labelled (with figure legend), and the presence of two additional sulcal plates is indicated by asterisks. q The two analysed strains cluster with sequences corresponding to type material of Kryptoperidinium triquetrum. www.nature.com/scientificreports/ guish between observational bias and true morphological differences among evolutionarily divergent clades of Kryptoperidinium.
The first detailed thecal pattern of Kryptoperidinium is based on material collected in the German Baltic Sea 55 , likely representing K. triquetrum (as Kryptoperidinium II has not been recorded from there so far). Anyhow, one of the schematic drawings of an apical view (later reproduced 97 ) differs significantly from all subsequent reports, namely in the symmetric and narrow plate 1′ having a central ventral position. This arrangement was rarely seen 55 and if so, then this 'Rautenplatte' was mostly fused with either plate 2′ (plate 1vap in E. Lindemann's notation) or with what was considered plate 1′′ (1pr in E. Lindemann's notation; note that E. Lindemann counted plates clockwise and thus different from the common Kofoidean notation). Such a 'fusion' in E. Lindemann's interpretation then leads to a large, asymmetric and slightly displaced ventral plate corresponding to our plate 1′ a. A symmetric, central, narrow plate 1′ was never observed in the present study and thus, the observation 55 should be taken with caution. Plates of Kryptoperidinium are thin and difficult to study, and it is possible that E. Lindemann erroneously interpreted artificially wrinkled plates dissembling the presence of a central, symmetric 'Rautenplatte' as a seeming indication of the close relationship between Kryptoperidinium and species of Peridinium. In any case, E. Lindemann's number of epithecal plates is (without a separate, narrow 'Rautenplatte') lower by 1 compared to the present (and other) observations, because he probably missed the narrow plate 7′′ (as inferred from his drawings).
One year after E. Lindemann's survey, three apical and seven precingular plates have been reported 94 . In this case, plate 1′ (in the present interpretation) was considered an element of the precingular plate series, and the correspondingly divergent plate pattern with (four apical and) only six precingular plates may result from neglecting again the narrow plate 7′′. The (mis-)interpretation of an apical as precingular plate has found its way into plate formulas provided in original literature 94 and also in seminal taxonomic compilations 97,[100][101][102][103] . They all specify three or four apical plates for K. triquetrum and thus create the impression of intraspecific variability regarding the plate numbers of the apical series. Considerable confusion also arose by the report of seven precingular though only three apical plates in cells from the Rio de Vigo estuary 53 . This Baiona strain is unfortunately lost, but another strain (VGO 1124) isolated from the same bloom (Isabel Bravo, pers. comm.) as well as strain VGO 556 from the nearby Ulla estuary clearly exhibit the usual plate pattern of K. triquetrum with four apical plates (Figs. S15, S16). Thus, the presence of three apical plates 53 is likely a misinterpretation due to difficulties to observe lateral sutures in this compressed species.
The same difficulty refers to unequivocal detection of lateral sutures of cingular plates and thus likely explains the report of six cingular plates 102 or of four cingular plates for the Baiona strain 53 . However, five cingular plates are clearly identified in the present material from the type locality as well as in the Spanish strains VGO 556 and VGO 1124, that they appear as correct and invariable number for K. triquetrum. This conclusion is also confirmed by other studies 43 , in one case even in combination with molecular data 54 , agreeing with the present sequences gained from the type material. Three strains of Kryptoperidinium II may have four cingular plates 52 . It cannot be excluded that the two clades of Kryptoperidinium differ by their number of cingular plates, but this needs confirmation by additional analyses of the plate patterns, particularly of strains assigned to Kryptoperidinium II.
For most species of dinophytes, number and arrangement of plates in the sulcal area are particularly difficult to ascertain. At a first glance, Kryptoperidinium appears easy to interpret, having three major sulcal plates forming a vertical row in the central ventral area 95 . The anterior plate is irregularly shaped and partly extends into the epitheca, and the interpretation as a sulcal plate 55 was followed by all subsequent authors ( Table 2). The present detailed analyses of sulcal plates, and the comparison of the ventral plate arrangement with other Kryptoperidiniaceae, allow for an alternative interpretation of this particular thecal element, which is usually labelled as anterior sulcal plate (Fig. 10) (Supplementary Figure 10). Particularly, the ventral view, and the sulcal plate arrangement of Durinskia oculata 32 , make an oblique split of an initially symmetric plate 1′ into an anterior (1′ a) and posterior part (1′ p) plausible for K. triquetrum (Fig. 10). This interpretation is supported by the unusually undulating course of the suggested split suture. Moreover, a very small and hook-shaped plate in the central sulcal area, adjacent to the area where the flagella emerge, conforms in shape and position with the anterior sulcal plate again of D. oculata (Fig. 10). This plate is interpreted here as anterior sulcal plate of Kryptoperidinium for the first time and has been already depicted but not labelled or discussed earlier (Figs. 2F 43 , 6D 54 ). Excluding plate 1′ p from the sulcal series, the present detailed analysis reveals the number of seven sulcal plates but because of the complex three-dimensional structure of the sulcal area with a tubular element in the centre, the small plates smp and sma are hard to detect in LM and are clearly identifiable by SEM only.   32 . In (B), a grey bar is hiding the undulating suture indicating that both plates likely belong to a large, symmetric first apical plate. Note that labelling of small sulcal plates in (C,D) differ, because K. triquetrum has two sulcal plates in median position (Fig. 4I). They are either absent from, or not detected yet (as they might be hidden behind the large Sd plate), for D. oculata. For DNA harvest, cells were collected by centrifugation (Eppendorf 5810R; Hamburg, Germany) in 50 mL centrifugation tubes at 3220×g for 10 min. Cell pellets were transferred with 0.5 mL lysis buffer (SL1, provided by the NucleoSpin Soil DNA extraction Kit; Macherey-Nagel; Düren, Germany) to 1 mL microtubes and stored frozen (− 20 °C) for subsequent DNA extraction.

Taxonomic activity
Microscopy. Observation of living or fixed cells (formaldehyde: 1% final concentration, or neutral Lugolfixed: 1% final concentration) was carried out using an inverted microscope (Axiovert 200 M; Zeiss; Munich, Germany) and a compound microscope (Axiovert 2; Zeiss), both equipped with epifluorescence and differential interference contrast optics. Living cells were recorded using a digital video camera (Gryphax, Jenoptik; Jena, Germany) at full-HD resolution. Single frame micrographs were extracted using Corel Video Studio software (Version X8 pro; Corel; Ottawa, Canada). Images of fixed cells were taken with a digital camera (Axiocam MRc5; Zeiss).
Light microscopic (LM) examination of thecal plates was performed on fixed cells (neutral Lugol) stained with Solophenyl Flavine (Carbosynth, Compton, UK), a fluorescent dye specific to cellulose 107 . Epifluorescence microscopy was used to observe chloroplasts (filter set 09; Zeiss) and to determine the shape and location of the nucleus (UV excitation, filter set 01; Zeiss) after staining of formalin-fixed cells with 4′,6-diamidino-2-phenylindole (DAPI, 0.1 μg mL −1 final concentration) for 10 min. Cell length and width were measured at × 1000 microscopic magnification using freshly fixed cells (formaldehyde, 1% final concentration) from dense but healthy and growing strains (based on stereomicroscopic inspection of the living material) at late exponential phase and the Axiovision software (Zeiss).
For scanning electron microscope (SEM), Lugol-fixed cells were collected by gentle filtration on 3 µm poresize polycarbonate filters and were subsequently processed for SEM (FEI Quanta FEG 200; Eindhoven, the Netherlands) as described previously 108 . Molecular phylogenetics. Genomic DNA was extracted following the manufacturers' instructions of the NucleoSpin Soil DNA extraction Kit (Macherey-Nagel, Düren, Germany) with an additional cell disruption step within the beat tubes; the samples were shaken in a FastPrep FP120 cell disrupter (Qbiogene, Carlsbad, USA-CA) for 45 s and another 30 s at a speed of 4.0 m s −1 . For the elution step, 50 μL of the provided elution buffer were spinned through the column, and elution was subsequently repeated with another 50 μL to increase the DNA yield. For the Kryptoperidinium host and for the endosymbiont, various regions of the ribosomal RNA (rRNA) were amplified using several primer sets (specific to dinophytes and their endosymbionts, respectively: Table S2) and temperature conditions (Table S3). Each reaction contained 16.3 μL of ultra-pure H 2 O, 2.0 μL of HotMaster Taq buffer (5Prime; Hamburg, Germany), 0.2 μL of each primer (10 μM), 0.2 μL of dNTPs (10 μM), 0.1 μL of Taq Polymerase (Quantabio; Beverly, USA-MA) and 1.0 μL of extracted DNA template (10 ng μL −1 ) to a final reaction volume of 20 μL. Afterwards, PCRs were conducted in a Nexus Gradient Mastercycler (Eppendorf), and PCR amplicons were inspected on a 1% agarose gel (in TE buffer, 70 mV, 30 min) to verify the expected length. If needed, nested PCR was performed with primer pairs indicated in Table S2. Chloroplast loci were amplified and sequences as described earlier 41 .
Amplicon purification followed the instructions of the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel), and PCR products were sequenced directly in both directions on an ABI PRISM 3730XL (Applied Biosystems; Waltham, USA-MA) using the ABI Big-Dye dye-terminator technique (Applied Biosystems) accordingly to the manufacturer's recommendations. Raw sequence data were processed using the CLC Genomics www.nature.com/scientificreports/ Workbench 12 (Qiagen; Hilden, Germany). Sequences were edited and assembled using Sequencher™v5.1 (Gene Codes; Ann Arbor, USA-MI). For visual comparison of the edited sequences, the alignment editor ′Se-Al′ (http:// tree. bio. ed. ac. uk/ softw are/ seal/) was used.
To compute a dinophyte reference tree inferred from a concatenated rRNA alignment 46,49 , we compiled a systematically representative set comprising 101 peridinialean dinophytes including 56 Kryptoperidiniaceae (Table S1). To compute a reference tree of Bacillariaceae inferred from a concatenated alignment comprising sequences of the rRNA operon, psbA, rbcL and psbC we used a previous alignment 41 and enriched the matrix with other relevant sequences 66 , also identified based on Blast searches 109 of the newly gained sequences from the endosymbionts. To build the alignment, separate matrices of the rRNA operon and the genes were constructed, aligned using 'MAFFT' v6.502a 110 , and the -qinsi option to take into account the secondary structure of rRNA, and concatenated afterwards. The aligned matrices are available in the Supplementary Information.
Phylogenetic analyses were carried out using Maximum Likelihood (ML) and Bayesian approaches, as described previously 91 , using the resources available from the CIPRES Science Gateway 111 . Briefly, the Bayesian analysis was performed using 'MrBayes' v3.2.7a 112 (freely available at http:// mrbay es. sourc eforge. net/ downl oad. php) under the GTR + Γ substitution model and the random-addition-sequence method with 10 replicates. We ran two independent analyses of four chains (one cold and three heated) with 20,000,000 generations, sampled every 1000th cycle, with an appropriate burn-in (10%) inferred from evaluation of the trace files using Tracer v1.7.1 113 . For the ML calculations, the MPI version of 'RAxML' v8.2.4 114 (freely available at http:// www. exeli xislab. org/) was applied using the GTR + Γ substitution model under the CAT approximation. We determined the best-scoring ML tree and performed 1000 non-parametric bootstrap replicates (rapid analysis) in a single step. The phylogenetic inferences were run in partitions under GTR (MrBayes) or in one block (RAxML, as it does not allow for empty sequences within partitions). Statistical support values (LBS: ML bootstrap support; BPP: Bayesian posterior probabilities) were drawn on the resulting, best-scoring tree.